Hanahan D, Lane D, Lipsich L, Wigler M, Botchan M
Cell. 1980 Aug;21(1):127-39. doi: 10.1016/0092-8674(80)90120-8.
A bacterial plasmid carrying the early region of SV40 (pOT) has been stably established in high molecular weight (hmw) DNA of mouse L cells by selection for the herpes virus thymidine kinase (tk) gene. DNA blotting has demonstrated that most cell lines contain multiple discrete copies of pOT, generally with an intact SV40 early region. No free copies of pOT have been detected. Both pOT and tk sequences may be amplified up to 20-200 copies of the SV40 early region. In contrast to the uniform staining pattern normally observed in SV40-transformed lines, indirect immunofluorescence using antiserum to the SV40 T antigen has demonstrated that the expression of the early region is heterogeneous in these cell lines. This fraction expressing T is characteristic of a given cell line, and varies from 0 to 99% positive. Several pOT cell lines have been fused to simian cells, and replicating low molecular weight DNAs were isolated from the heterokaryons. Transformation of E. coli with this DNA demonstrates that pOT can be rescued from hmw DNA in L cells and reestablished as a plasmid in E. coli. Excision is generally precise when pOT is introduced to the murine cells as supercoiled molecule, and imprecise when pOT is introduced in linear form.
通过选择疱疹病毒胸苷激酶（tk）基因，携带SV40早期区域的细菌质粒（pOT）已稳定整合到小鼠L细胞的高分子量（hmw）DNA中。DNA印迹分析表明，大多数细胞系含有多个离散的pOT拷贝，通常具有完整的SV40早期区域。未检测到游离的pOT拷贝。pOT和tk序列均可扩增至SV40早期区域的20 - 200个拷贝。与通常在SV40转化细胞系中观察到的均匀染色模式不同，使用抗SV40 T抗原血清进行的间接免疫荧光显示，这些细胞系中早期区域的表达是异质性的。表达T抗原的细胞比例是特定细胞系的特征，从0%到99%呈阳性变化。已将几个pOT细胞系与猿猴细胞融合，并从异核体中分离出正在复制的低分子量DNA。用这种DNA转化大肠杆菌表明，pOT可以从L细胞的hmw DNA中拯救出来，并在大肠杆菌中重新构建为质粒。当pOT以超螺旋分子形式导入小鼠细胞时，切除通常是精确的；而当pOT以线性形式导入时，切除则是不精确的。
